Basanta-Sanchez Maria, Temple Sally, Ansari Suraiya A, D'Amico Anna, Agris Paul F
The RNA Institute, University at Albany, Albany, NY, USA
Neural Stem Cell Institute, Rensselaer, NY, USA.
Nucleic Acids Res. 2016 Feb 18;44(3):e26. doi: 10.1093/nar/gkv971. Epub 2015 Oct 4.
Exploration of the epitranscriptome requires the development of highly sensitive and accurate technologies in order to elucidate the contributions of the more than 100 RNA modifications to cell processes. A highly sensitive and accurate ultra-high performance liquid chromatography-tandem mass spectrometry method was developed to simultaneously detect and quantify 28 modified and four major nucleosides in less than 20 min. Absolute concentrations were calculated using extinction coefficients of each of the RNA modifications studied. A comprehensive RNA modifications database of UV profiles and extinction coefficient is reported within a 2.3-5.2 % relative standard deviation. Excellent linearity was observed 0.99227-0.99999 and limit of detection values ranged from 63.75 attomoles to 1.21 femtomoles. The analytical performance was evaluated by analyzing RNA modifications from 100 ng of RNA from human pluripotent stem cell-derived neural cells. Modifications were detected at concentrations four orders of magnitude lower than the corresponding parental nucleosides, and as low as 23.01 femtograms, 64.09 attomoles. Direct and global quantitative analysis of RNA modifications are among the advantages of this new approach.
对表观转录组的探索需要开发高度灵敏和准确的技术，以阐明100多种RNA修饰对细胞过程的作用。我们开发了一种高度灵敏且准确的超高效液相色谱 - 串联质谱方法，可在不到20分钟的时间内同时检测和定量28种修饰核苷和4种主要核苷。使用所研究的每种RNA修饰的消光系数计算绝对浓度。报告了一个包含紫外光谱和消光系数的全面RNA修饰数据库，相对标准偏差在2.3 - 5.2%范围内。观察到良好的线性关系，相关系数为0.99227 - 0.99999，检测限范围为63.75阿托摩尔至1.21飞摩尔。通过分析来自人多能干细胞衍生神经细胞的100 ng RNA中的RNA修饰来评估分析性能。检测到的修饰浓度比相应的亲本核苷低四个数量级，低至23.01飞克，64.09阿托摩尔。直接和全局定量分析RNA修饰是这种新方法的优点之一。
